Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism

Abstract

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.Cells. Murine NIH 3T3 fibroblasts (American Type Culture Collection [ATCC] CRL1658; ATCC, Rockville, Md.) were propagated in Dulbecco's modified essential medium (DMEM; Mediatech, Herndon, Va.) supplemented with 10% heat-inactivated bovine calf serum (HyClone Laboratories, Logan, Utah) and 1% L-glutamine (Gibco/BRL, Grand Island, N.Y.). IC-21 cells, a simian virus 40-transformed peritoneal macrophage cell line derived from C57BL/6 mice (41), were obtained from ATCC and were propagated in RPMI medium (Mediatech) supplemented with 10% heat-inactivated fetal calf serum (Gibco/BRL) and 1% L-glutamine. Primary fibroblasts, derived from the embryos of timedpregnant BALB/c.ByJ mice (Jackson Laboratory, Bar Harbor, Maine) on day 19 of gestation, were cultured in DMEM containing 20% heat-inactivated fetal calf serum, 1% L-glutamine, and 50 g of gentamicin sulfate (Sigma Chemical Co., St. Louis, Mo.) per ml.Virus. The parental WT MCMV used in this study was of the Smith strain (ATCC VR-194). Stocks of WT and mutant viruses were prepared in and titers were determined on NIH 3T3 cells as previously described (8). Mock preparations of virus consisted of culture supernatants from noninfected NIH 3T3 cells.Mice. BALB/c.ByJ mice, either 20-day-old weanlings (...

Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control

“…The fcr-1 gene is nonessential for replication in cell culture and has no phenotype in vitro. Herpesvirus functions have been shown to modulate virus-host interactions such as antigen presentation and immune system control (2,4,12,15,17,31,34,37,(39)(40)(41), tissue tropism (3-5, 23, 27), virus spread (9,25,42), and the establishment of latency (33). Most of these functions are encoded by nonessential genes.…”

“…In view of our results, the reduced in vivo virulence of these recombinants can now be explained at least in part by the lack of the fcr-1 gene. It has been shown that the deletion mutant missing both the fcr-1 gene and the m137 gene grew normally in the IC-21 macrophage cell line (4). This indicates that the FcR acts differently from the neighboring genes M140 and/or M141, to which macrophages growth deficiency was mapped.…”

Transfusion‐transmitted cytomegalovirus (CMV) infections in a murine model: characterization of CMV‐infected donor mice

“…Resulting samples were serially diluted and plated in replicates on 96-well plates containing NIH/3T3 monolayers, which are permissive for MCMV replication. 28 Monolayers were monitored daily for up to 42 days. Based on visual detection of viral cytopathic effect, each well was scored as positive or negative.…”

The pathogenicity of cytomegalovirus

“…Deletion of several MCMV non‐essential genes has proven to affect tissue tropism in vivo. For example, a recombinant virus (RV7) with a 7.7‐kb deletion spanning portions of the HCMV Hin dIII‐J and ‐I region has shown that genes m137, m138, M139, M140 and M141 are not essential for virus replication in vitro but are essential for virus replication in vivo [73–75]. The functions of these genes are largely unknown.…”